Not applicable.
Transplantation of cells exhibiting glucose-responsive insulin secretion has the potential to cure diabetes. However, this approach is limited by an inadequate supply of cells with that property, with is exhibited only by pancreatic xcex2-cells. The development of expanded populations of human xcex2-cells that can be used for cell transplantation is therefore a major goal of diabetes research (D. R. W. Group, xe2x80x9cConquering diabetes: a strategic plan for the 21st centuryxe2x80x9d NIH Publication No . 99-4398 (National Institutes of Health, 1999)). A number of alternative approaches are being pursued to achieve that goal, including using porcine tissue as a xenograft (Groth et al., J Mol Med 77:153-4 (1999)), expansion of primary human xcex2-cells with growth factors and extracellular matrix (Beattie et al., Diabetes 48:1013-9 (1999)), and generation of immortalized cell lines that exhibit glucose-responsive insulin secretion (Levine, Diabetes/Metabolism Reviews 1:209-46 (1997)).
Although there has been great interest in using porcine islets, they are difficult to manipulate in vitro and concerns have been raised about endogenous and exogenous xenobiotic viruses being transmitted to graft recipients (Weiss, Nature 391:327-8 (1998)). With primary human xcex2-cells, entry into the cell cycle can be achieved using hepatocyte growth factor/scatter factor (xe2x80x9cHGF/SFxe2x80x9d) plus extracellular matrix (xe2x80x9cECMxe2x80x9d) (Beattie et al., Diabetes 48:1013-9 (1999), Hayek et al., Diabetes 44:1458-1460 (1995)). However, this combination, while resulting in a 2-3xc3x97104-fold expansion in the number of cells, is limited by cellular senescence and loss of differentiated function, particularly pancreatic hormone expression (Beattie et al., Diabetes 48:1013-9 (1999)).
Immortalized cell lines from the human endocrine pancreas have been created to develop xcex2-cell lines that exhibit glucose responsive insulin secretion (Wang et al., Cell Transplantation 6:59-67 (1997), Wang et al., Transplantation Proceedings 29:2219 (1997), Halvorsen et al., Molecular and Cellular Biology 19:1864-1870(1999)). The cell lines are made by infecting primary cultures of cells from various sources including adult islets, fetal islets, and purified xcex2-cells, with viral vectors expressing the potent dominant oncogenes such as SV40 T antigen and H-rasval12(Wang et al., Cell Transplantation 6:59-67 (1997), Wang et al., Transplantation Proceedings 29:2219(1997), Halvorsen et al., Molecular and Cellular Biology 19:1864-1870 (1999); see also U.S. Pat. No. 5,723,333). The combined effect of those oncogenes is to trigger growth factor-independent and extracellular matrix (ECM)-independent entry into the cell cycle, as well as to prolong the lifespan of the cells from 10-15 population doublings or primary cells to approximately 150 doubling for the oncogene-expressing cells (Halvorsen et al., Molecular and Cellular Biology 19:1864-1870 (1999)). Further introduction of the gene encoding the hTRT component of telomerase results in immortalization, allowing the cells to be grown indefinitely (Halvorsen et al., Molecular and Cellular Biology 19:1864-1870 (1999)).
Although the cell lines grow indefinitely, they lose differentiated function, similar to growth-stimulated primary xcex2-cells. Methods of stimulating differentiation of the cell lines into insulin-secreting xcex2-cells are therefore desired. Such cells could then be transplanted in vivo as a treatment for diabetes.
Induction of xcex2-cell differentiation in cultured human xcex2-cells was achieved by stimulating multiple signaling pathways, including those downstream of the homeodomain transcription factor PDX-1, cell-cell contact, and the glucagon-like peptide-1 (GLP-1) receptor. Synergistic activation of those pathways resulted in differentiation of the cultured human xcex2-cells, which initially express no detectable pancreatic hormones, into fully functional xcex2-cells that exhibit glucose-responsive insulin secretion. Furthermore, these cells can be transplanted in vivo and demonstrate glucose-responsive expression of insulin. The ability to grow unlimited quantities of functional human xcex2-cells in vitro provides the means for a definitive cell transplantation therapy for treatment of diabetes.
In one aspect, the present invention provides a method for inducing insulin gene expression in cultured endocrine pancreas cells, the method comprising the steps of (i) expressing a PDX-1 gene in cells that have been cultured under conditions such that the cells are in contact with other cells in the culture; and (ii) contacting the cells with a GLP-1 receptor agonist, thereby inducing insulin gene expression in the cells.
In another aspect, the present invention provides a method of identifying a compound that modulates xcex2-cells function, the method comprising the steps of contacting cells made by the method described above with the compound and determining the effect of the compound on xcex2-cell function.
In another aspect, the present invention provides a stable culture of endocrine pancreas cells, wherein the cells are in contact with other cells in the culture, wherein the cells express a PDX-1 gene, and wherein insulin gene expression is stimulated in the cells when exposed to an effective amount of a GLP-1 receptor agonist.
In another aspect, the present invention provides a method of treating a diabetic subject by providing to the subject cells that secrete insulin in response to glucose, the method comprising the steps of: (i) contacting a culture of endocrine pancreas cells expressing a PDX gene with a GLP-1 receptor agonist, wherein the cells have been cultured under conditions such that the cells are in contact with other cells in the culture; and (ii) administering the cells to the subject, thereby providing to the subject cells that secrete insulin in response to glucose.
In one embodiment, the GLP-1 receptor agonist is a GLP-1 analog or has an amino acid sequence of a naturally occurring peptide. In another embodiment, the GLP-1 receptor agonist is GLP-1, exendin-3, or exendin-4.
In one embodiment, the cells are cultured as aggregates in suspension.
In one embodiment, the PDX-1 gene is endogenous to the cells. In another embodiment, the PDX-1 gene is recombinant.
In one embodiment, the cells are human cells. In another embodiment, the cells are xcex2lox5 cells. A deposit of the xcex2lox5 cells, which is a cell line of human pancreatic cells, was made on Jul. 19, 2001 under accession number PTA-3532 at the American Type Culture Collection, 10801 University Boulevard, Manassas, Va. 20110-2209.
In one embodiment, the cells express a recombinant oncogene. In another embodiment, the cells express a recombinant oncogene. In another embodiment, the cells express a recombinant oncogene. In another embodiment, the cells express a recombinant telomerase gene.
In one embodiment, the diabetic subject is a human. In another embodiment, the subject has Type I insulin dependent diabetes.